To date, there is no representative in vitro model for liver sinusoidal endothelial cells (LSECs), as primary LSECs dedifferentiate very fast in culture and no combination of cytokines or growth factors can induce an LSEC fate in (pluripotent stem cell (PSC)-derived) endothelial cells (ECs). Furthermore, the transcriptional programmes driving an LSEC fate have not yet been described. Here, we first present a computational workflow (CenTFinder) that can identify transcription factors (TFs) that are crucial for modulating pathways involved in cell lineage specification. Using CenTFinder, we identified several novel LSEC-specific protein markers, such as FCN2 and FCN3, which were validated by analysis of previously published single-cell RNAseq data. We also identified PU.1 (encoded by the SPI1 gene) as a major regulator of LSEC-specific immune functions. We show that SPI1 overexpression (combined with the general EC TF ETV2) in human PSCs induces ECs with an LSEC-like phenotype. The ETV2-SPI1-ECs display increased expression of LSEC markers, such as CD32B and MRC1, as well as several of the proposed novel markers. More importantly, ETV2-SPI1-ECs acquire LSEC functions, including uptake of FSA-FITC, as well as labelled IgG. In conclusion, we present the CenTFinder computational tool to identify key regulatory TFs within specific pathways, in this work pathways of lineage specification, and we demonstrate its use by the identification and validation of PU.1 as a master regulator for LSEC fating.

迄今为止，肝窦内皮细胞 (LSEC) 还没有具有代表性的体外模型，因为原代 LSEC 在培养中去分化非常快，并且细胞因子或生长因子的组合不能诱导（多能干细胞 (PSC) 衍生的 LSEC 命运）。 ) 内皮细胞 (EC)。此外，尚未描述驱动 LSEC 命运的转录程序。在这里，我们首先提出一个计算工作流程 ( CenTFinder )，它可以识别转录因子 (TF)，这些转录因子对调节细胞谱系规范所涉及的通路至关重要。使用CentFinder，我们确定了几种新的 LSEC 特异性蛋白质标记物，例如 FCN2 和 FCN3，这些标记物通过对先前发表的单细胞 RNAseq 数据的分析得到验证。我们还确定 PU.1（由SPI1基因编码）是 LSEC 特异性免疫功能的主要调节因子。我们表明人类 PSC中的SPI1过表达（与一般 EC TF ETV2 结合）诱导具有 LSEC 样表型的 EC。ETV2-SPI1-ECs 显示 LSEC 标记物（如 CD32B 和 MRC1）以及一些提议的新型标记物的表达增加。更重要的是，ETV2-SPI1-ECs 获得 LSEC 功能，包括摄取 FSA-FITC，以及标记的 IgG。总之，我们介绍了CentFinder 在谱系规范的这项工作途径中识别特定途径中的关键调节 TF 的计算工具，我们通过识别和验证 PU.1 作为 LSEC 命运的主调节器来证明其用途。